Search PubMed⌕ Search

Biomedical subjects

R R Rando

Publications and source records attributed to R R Rando.

At least 109 records · Page 6Linked to original sources
PubMed1987

Isomerization of all-trans-retinoids to 11-cis-retinoids in vitro.

The key biochemical process of the vertebrate visual cycle required for rhodopsin regeneration, 11-cis-retinoid production from all-trans-retinoids, is shown to occur in vitro. A 600 X g supernatant from a frog retina/pigment epithelium homogenate transforms added all-trans-[3H]retinol, in a time-dependent fashion, to a mixture of 11-cis-retinol, 11-cis-retinal, and 11-cis-retinyl palmitate. 13-cis-Retinoids are formed in only minor amounts by nonspecific processes. Studies using washed particulate fractions of the 600 X g supernatant indicate that all-trans-[3H]retinol is isomerized to 11-cis-retinoids much more effectively than is all-trans-[3H]retinal or all-trans-[3H]retinyl palmitate. The 11-cis-retinoid biosynthetic activity is heat-labile, sedimentable by high-speed centrifugation, and largely found in the pigment epithelium rather than in the neural retina.

Animals↗
PubMed1986

In vivo isomerization of all-trans- to 11-cis-retinoids in the eye occurs at the alcohol oxidation state.

The vertebrate biochemical pathway for regeneration of visual pigments in the living eye after bleaching is largely uncharacterized. Since isomerization of an all-trans-retinoid to an 11-cis-retinoid could conceivably occur via the aldehyde, alcohol, or ester forms of vitamin A, it is important to determine the oxidation state of the retinoid that is isomerized in vivo. To address this problem, light-adapted rats and frogs were injected intraperitoneally with a mixture of [15-3H]-all-trans-retinol and [15-14C]-all-trans-retinol. After 4 or 24 h of dark adaptation, labeled retinoids in the animal's eyes were analyzed. All rats had the expected 50% loss of 3H label (relative to 14C) in 11-cis-retinal, a loss of 3H that must occur when [15-3H]retinol is oxidized to retinal. 11-cis-Retinyl esters in the rats' eyes at 4 h retained 67% of the 3H label, and this could be increased to 81% when the rats were pretreated with 4-methylpyrazole, an alcohol dehydrogenase inhibitor known to inhibit dark adaptation. This result demonstrates that retinoid isomerization occurs at the alcohol oxidation state in the rat eye. Had it occurred at the aldehyde oxidation state, at least 50% of the 3H in the 11-cis-retinyl esters would have been lost. The importance of this isomerization pathway is emphasized by the observation that dark-adapting rats whose alcohol dehydrogenase(s) had been inhibited by 4-methylpyrazole had increased amounts of 11-cis-retinyl ester in their eyes relative to control rat eyes, a result that is understandable only if retinoids are isomerized in vivo at the alcohol oxidation state.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Chemical modification of rhodopsin and its effect on regeneration and G protein activation.

The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Mechanism of action of aromatic amines that short-circuit the visual cycle.

DAPP [1,5-bis(p-aminophenoxy)pentane] is an antischistosomal drug that can inhibit dark adaptation in vertebrates by impairing formation of 11-cis-retinoids in the eye and by depleting preformed stores of them [Bernstein, P. S., & Rando, R. R. (1985) Vis. Res. 25, 741-748]. It has recently been shown that p-phenetidine and other monofunctional analogues of DAPP (a symmetric bifunctional molecule) can duplicate DAPP's effects, and it was proposed that these retinotoxic compounds exert their effects in vivo by "short-circuiting" the visual cycle, catalyzing the thermodynamically downhill isomerization of 11-cis-retinal to all-trans-retinal [Bernstein, P. S., Lichtman, J. R., & Rando, R. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1632-1635]. In this paper, the "short-circuit" hypothesis is investigated more fully. Numerous phenetidine-like molecules are assayed for their ability to inhibit rhodopsin formation and 11-cis-retinyl palmitate formation in the living frog eye. It is found that virtually any aromatic amine with a moderately hydrophobic alkyl chain "tail" is an active inhibitor in vivo. The tail can be in either the para or the meta position and can be attached to the aromatic ring either by direct linkage or by an ether linkage. Compounds that can be metabolized in vivo to such active compounds are also inhibitory. Amino group modification studies demonstrate an absolute requirement for structures that can form a Schiff base with retinal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Ocular↗
PubMed1986

Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein.

Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction.

Animals↗
PubMed1986

Short-circuiting the visual cycle with retinotoxic aromatic amines.

The retinotoxic drug 1,5-di-(p-aminophenoxy)pentane inhibits the accumulation of all 11-cis-retinoids in the eye and can deplete preformed stores of them. It is shown here that these effects are not specific to 1,5-di-(p-aminophenoxy)pentane but are shared generally by primary aromatic amines containing a hydrophobic tail. Furthermore, certain clinically used drugs, such as the anti-inflammatory drug phenacetin, can be metabolized to produce these retinotoxic amines. It is likely that hydrophobic aromatic amines will in general be retinotoxic, and drugs based on these structures need to be reassessed in this light. It is proposed here that these amines function by catalyzing the isomerization of 11-cis-retinal thermodynamically downhill to form its all-trans congener. This mechanism accounts for the lack of structural specificity observed with these compounds and is supported by experimental evidence presented here. Schiff bases formed between 11-cis-retinal and a relevant aromatic amine in phosphatidylcholine-based liposomes lead to the formation of the all-trans isomer, at rates approximately equal to 15 times faster than the rate of 11-cis-retinal isomerization by itself in these liposomes and 10(2)-10(3) times faster than the rate of isomerization of this molecule in n-heptane. The rates of the amine-catalyzed isomerization are fast enough to account for their in vivo effect.

Amines↗
PubMed1985

Methylation of the active-site lysine of rhodopsin.

Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

all-trans-retinoids and dihydroretinoids as probes of the role of chromophore structure in rhodopsin activation.

The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8-dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment.

Animals↗
PubMed1985

The nature of protein kinase C activation by physically defined phospholipid vesicles and diacylglycerols.

Protein kinase C is activated by a 1,2-sn-diacylglycerol and phospholipid at low calcium concentrations. Of the various phospholipids studied, phosphatidylserine has been shown to be the most effective one and is usually used in assaying the enzyme (Kaibuchi, K., Takai, Y., and Nishizuka, Y. (1981) J. Biol. Chem. 256, 7146-7149). It is shown here that under the conditions of the enzymatic assay, phosphatidylserine does not form typical fluid bilayer structures as seen by electron microscopy and fluorescence polarization. On the other hand, 1:4 phosphatidylserine/phosphatidylcholine bilayer vesicles can be formed which support protein kinase C activation. They have the advantage in that they are characterizable, form physiologically relevant bilayer structures, and are readily and reproducibly formed. In addition, they do not support protein kinase C activation in the absence of added diacylglycerol, a property that makes them invaluable in studying the role of diacylglycerol structure in protein kinase C activation. It is further demonstrated that the rat brain enzyme is activated by 1,2-sn-diolein but not by 2,3-sn-diolein nor 1,3-diolein, demonstrating the high specificity of the kinase toward the glycerol backbone. 1,2-rac-Dielaidin, 1,2-rac-distearin, and 1,2-sn-dipalmitin are all active, which is consistent with the idea that the specificity of protein kinase C is not directed toward the fatty acid side chain of the diacylglycerols.

Animals↗
PubMed1985

Biochemical properties of 9-cis- and all-trans-retinoylopsins.

The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.

Animals↗
PubMed1985

Nonstereospecific biosynthesis of 11-cis-retinal in the eye.

[3H]-all-trans-Retinol injected intraocularly into rats is processed to [3H]-11-cis-retinal, the visually active retinoid that binds to opsin. After 18 h, virtually all (93%) of the radioactive retinals recovered were in the form of 11-cis-retinal. At earlier times, however, both all-trans- and 13-cis-retinals, the latter being a nonphysiological isomer, were formed. Both of these isomers disappeared concomitant with the formation of 11-cis-retinal. The rise and fall of 13-cis-retinal suggest that this isomer can be converted into 11-cis-retinal either directly or indirectly in vivo and, hence, that the biosynthesis of the latter is nonstereospecific. This hypothesis was verified by showing that in double-labeling experiments [14C]-13-cis-retinol was converted into 11-cis-retinal nearly as well (approximately 70%) as [3H]-all-trans-retinol. These studies show that the biosynthesis of 11-cis-retinal can be nonstereospecific and, hence, that the process may be chemically rather than enzymatically mediated in vivo. In contrast, double-labeling studies with [14C]-9-cis-retinol and [3H]-all-trans-retinol showed that very little, if any, of the 9-cis isomer was processed to 11-cis-retinal in vivo although it did form isorhodopsin. This is consistent with what is known about the relative chemical stabilities of 9-cis-retinoids from model studies. The isomerization of 9-cis-retinoids is much slower than that of their all-trans, 13-cis, or 11-cis congeners. These results are discussed in terms of a possible mechanism for the biosynthesis of 11-cis-retinal in vivo and suggest that the isomerization event need not necessarily be enzyme mediated.

Animals↗
PubMed1985

The specific inhibition of 11-cis-retinyl palmitate formation in the frog eye by diaminophenoxypentane, an inhibitor of rhodopsin regeneration.

The antischistosomal drug 1,5-di-(p-aminophenoxy) pentane (DAPP), an inhibitor of rhodopsin regeneration in the vertebrate retina, is shown to completely block the production of 11-cis-retinyl palmitate in the frog eye. An untreated frog generates a large amount of 11-cis-retinyl palmitate during 1-2 days in the dark after a strong bleach. Also, it is demonstrated that DAPP can deplete the stores of 11-cis-retinyl palmitate in the dark-adapted frog eye. The specificity of DAPP's inhibition of dark-adaptation is explored, and the usefulness of employing retinotoxic drugs to investigate the physiology and biochemistry of rhodopsin regeneration is discussed.

Aniline Compounds↗
PubMed1984

The stereospecific activation of protein kinase C.

Protein kinase C is synergistically activated by the presence of calcium, certain phospholipids and a diacylglycerol. The physiological activation of the enzyme appears to be determined by the availability of the diacylglycerol which is itself a product of (poly) phosphoinositol turnover. It is shown here that the diacylglycerol activation effect is stereospecific, with only the 1,2-sn-diglycerides being active. This demonstrates for the first time a stereospecific effector role for a membrane-bound lipid. Furthermore, this work strengthens the link forged between the highly potent and specific tumor promoters (such as the phorbol esters) and the diglycerides as activators of protein kinase C.

Animals↗
PubMed1984

Lateral mobility of phospholipid and cholesterol in the human erythrocyte membrane: effects of protein-lipid interactions.

The phospholipid and cholesterol derivatives N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N1-cholesterylcarbamoyl-N8-(7-nitro-2,1,3-benzoxadiazol-4-yl )-3,6-dioxaoctane-1 , 8-diamine (NBD-Chol), respectively, were incorporated into egg phosphatidylcholine/cholesterol multilamellar liposomes, human erythrocyte ghost membranes, and multilamellar liposomes derived from extracted human erythrocyte membrane lipids. The lateral mobility of these probes in the plane of the various membranes was measured by using the fluorescence photo-bleaching recovery technique. NBD-PE and NBD-Chol manifested identical lateral mobilities in egg phosphatidylcholine/cholesterol multilamellar liposomes over the range of temperatures from 10 to 37 degrees C and the range of cholesterol mole fractions from 0.0 to 0.5, and in erythrocyte ghost membranes and erythrocyte membrane lipid-derived multilamellar liposomes over the range of temperatures from 15 to 37 degrees C. The weak temperature dependence of the lateral diffusion coefficients of the lipid probes in both artificial and erythrocyte ghost membranes is consistent with the lack of a phase transition in any of these systems over the temperature range studied. Both NBD-PE and NBD-Chol diffuse 4-fold faster in liposomes derived from extracted erythrocyte membrane lipids (D = 8.0 X 10(-9) cm2 s-1 at 37 degrees C) than in the ghost membranes themselves (D = 2.1 X 10(-9) cm2 s-1 at 37 degrees C), suggesting a significant restriction of lipid lateral mobility by membrane protein in the human erythrocyte membrane.

Cholesterol↗
PubMed1984

Specific reaction of 9-cis-retinoyl fluoride with bovine opsin.

Opsin readily undergoes Schiff base formation between an active site lysine and 9-cis- or 11-cis-retinaldehyde to form the visual pigments isorhodopsin (lambda max = 487 nm) and rhodopsin (lambda max = 500 nm), respectively (Dratz, 1977). It would be predicted that 9-cis-retinoyl fluoride (1), an isostere of 9-cis-retinal, should be an active site directed, mechanism-based labeling agent of opsin, since a stable peptide bond should be formed instead of a Schiff base. It is shown here that 9-cis-retinoyl fluoride (1) reacts with opsin in a time-dependent fashion (t1/2 = 9 min at 25 microM 1) to form a new, nonbleachable pigment with a lambda max of approximately 365 nm. beta-Ionone competitively slows down the rate of the reaction. The absorbance of the new pigment at approximately 365 nm is similar to that of model amide compounds. This result is consistent in a general and qualitative way with the Nakanishi-Honig point-charge model for visual pigments which requires that the chromophore be charged, a situation not possible when the retinoid is linked to opsin via a peptide bond rather than a protonated Schiff base [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A., Arnabaldi, M., & Motto, M.G. (1979) J. Am. Chem. Soc. 101, 7084-7086]. 9-cis-Retinoyl fluoride (1) is approximately 4-fold more potent than all-trans-retinoyl fluoride (2) as an inactivator of bovine opsin. Importantly, 13-cis-retinoyl fluoride (3) is inactive, and no new absorption band at 365 nm is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Ontogenesis of dopaminergic neurons in the post-natal rabbit retina: pre- and post-synaptic elements.

The post-natal development of several aspects of the dopaminergic system in the rabbit have been examined. Dopamine (DA) itself was barely detectable from soon after birth up to about 7 days of age. Thereafter, dramatic increases in the retinal content of DA and also of the two major enzymes involved in its biosynthesis were observed, approaching adult values by about 16 days. These changes occur when functional synapses are being formed as judged by electrophysiological and anatomical studies. On the other hand, the post-synaptic target of these neurons, DA-stimulated adenylate cyclase could be easily detected at 3 days of age and did not appear to undergo any dramatic changes in activity during development. The affinity of haloperidol for this receptor did, however, appear to decrease with age suggesting that the receptor is 'born' in a supersensitive state but attains 'normal' properties after the onset of DA neurotransmission. Depletion of DA stores during these early stages by irreversible inhibition of biosynthesis in vivo did not appear to halt the development of tyrosine hydroxylase or have any irrevocable effects on DA neurons. This suggests that the continued presence of neurotransmitter is not necessary for the developmental process to continue that the commitment of particular neurons to become dopaminergic may be made earlier than 5-6 days post-natally.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1983

Effects of light on dopamine metabolism in the chick retina.

The effect of prolonged exposure to light on the activity of dopaminergic neurons and dopamine (DA) metabolism of chick retinae was investigated. alpha-Fluoromethyldopa, a potent and specific irreversible inactivator of aromatic amino acid decarboxylase, was used to assess DA turnover after inhibition of synthesis, and also to assess in vivo tyrosine hydroxylase activity by dihydroxyphenylalanine accumulation. After 48 h of light exposure, retinal DNA in 12-day-old chicks was about 30% higher (p less than 0.005) whereas dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated two to three times (p less than 0.005) the level of controls kept in the dark for the same period. DA turnover was about twofold faster in the light (t 1/2 = 31 min) than in the dark (t 1/2 = 65 min). Tyrosine hydroxylase, assayed in vitro with saturating levels of cofactor and substrate, increased by about 50% after light exposure. The apparent tyrosine hydroxylase activity in vivo was approximately sixfold higher in the light than the dark. These results are interpreted and discussed in terms of the regulation of DA synthesis, and the use of DOPAC and HVA as indices of DA function in the retina.

3,4-Dihydroxyphenylacetic Acid↗